WO1997004810A1 - Proteine dax-1, procedes de production et d'utilisation - Google Patents

Proteine dax-1, procedes de production et d'utilisation Download PDF

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WO1997004810A1
WO1997004810A1 PCT/US1996/012310 US9612310W WO9704810A1 WO 1997004810 A1 WO1997004810 A1 WO 1997004810A1 US 9612310 W US9612310 W US 9612310W WO 9704810 A1 WO9704810 A1 WO 9704810A1
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dax
gene
protein
vector
dna
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PCT/US1996/012310
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WO1997004810A9 (fr
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Edward R. B. Mccabe
Weiwen Guo
Thomas P. Burris
Eric Vilain
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The Regents Of The University Of California
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Priority to AU66010/96A priority Critical patent/AU6601096A/en
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Publication of WO1997004810A9 publication Critical patent/WO1997004810A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C07K14/721—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70567—Nuclear receptors, e.g. retinoic acid receptor [RAR], RXR, nuclear orphan receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • AHC is an inherited disorder of adrenal gland development. The disorder results in adrenal insufficiency early in infancy, with low serum concentration of glucocorticoids, mineralocorticoids and androgens, and failure to respond to ACTH
  • AHC is a rare disorder with an overall estimated frequency of 1:12,500 live births.
  • Clinical signs and symptoms of infants with adrenal hypoplasia include poor '? feeding, failure to gain weight, hyperpigmentation, vomiting, diarrhea, vascular collapse, and sudden death. Dehydration, hyponatremia, hyperkalemia, acidosis,
  • HH is commonly associated with the X-linked form of the disease, and is generally noted at the expected time of puberty. It is not clear if this form of HH is of pituitary or hypothalamic origin. HH often is noted in boys with X-linked AHC at the expected time of pubertal maturation. Furthermore, abnormalities of the genitourinary system appear to occur with increased frequency in boys with AHC, and these include: cryptorchidism, hypospadias, small external genitalia, ureteral reflux, and urethral stenosis.
  • the nuclear hormone receptor superfamily are ligand dependent transcription factors which modulate a large number of essential cellular processes. Members of the superfamily regulate a myriad of pathways in higher organisms ranging from development and morphogenesis to reproduction, behavior and homeostasis.
  • the superfamily consists of receptors for steroid hormones (e.g. corticosteroids, estrogens, progestins, and androgens), steroid derivatives (dihydroxy vitamin D3) and non-steroids (thyroid hormone and retinoids).
  • steroid hormones e.g. corticosteroids, estrogens, progestins, and androgens
  • steroid derivatives dihydroxy vitamin D3
  • non-steroids thyroid hormone and retinoids
  • Nuclear hormone receptors are localized within a cell and, in contrast to receptors for peptide hormones, are not cell-membrane associated. They mediate their function in the cell nucleus by directly influencing gene expression.
  • the nuclear hormone receptors have high affinity for their ligand which is in the range of 10-9 to 10-11 M. It is believed that the lipophilic hormone passes through the cell membrane without the help of specialized membrane bound accessory proteins and binds to the receptor within the cell. Hormone binding has a drastic effect in transforming the receptor by changing its conformation and thereby altering its function and activity.
  • One such group of hormones is the corticosteroids, which can be divided into glucocorticoids (cortisol and corticosterone) and mineralocorticoids (aldosterone). They are synthesized in the adrenal cortex using cholesterol as a precursor. Also synthesized in the adrenal cortex are androgens, which affect growth, development of skeletal muscle and behavior.
  • DAX-1 protein is a novel member of the nuclear hormone receptor superfamily and may bind a retinoic acid or other response element.
  • DAX-1 is expressed in steroidogenic cells in mammals including the adrenal gland, ovaries, testes, hypothalamus and pituitary gland.
  • the DAX-1 protein has the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in Figure 12.
  • a DAX-1 protein molecule expressed by species other than the human species is encompassed within this invention. It will share substantial homology with the DAX-1 protein having the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in Figure 12.
  • the data herein support the existence of the DAX-1 protein expressed by various species, e.g., from mammals to yeast ( Figure 10). A homologous sequence has been found also in CL. elegans.
  • the invention also provides nucleic acid molecules encoding the protein of the invention ( Figure 12).
  • the nucleic acid molecule is a DNA molecule.
  • the genomic DNA molecule includes both the intron and exons of the sequence which encodes DAX-1.
  • the nucleic acid molecule is a cDNA molecule as shown in Figure 12.
  • the proteins and nucleic acid molecules of the present invention are weapons that can be used as part of an arsenal of weapons against adrenocortical disorders of metabolism and development.
  • the invention also provides methods of gene therapy which utilize DAX-1 nucleic acid moleules.
  • a gene encoding a DAX-1 protein is transferred into steroidogenic cells.
  • a vector comprising DNA encoding a DAX-1 protein is introduced into a producer cell which results in the integration of the vector into the cell.
  • Producer cells into which the vector is integrated are then selected.
  • the producer cell is grafted proximate to the steroidogenic cells so that the steroidogenic cells are infected with the vector produced by the producer cell and the gene encoding DAX- 1 is transferred to the steroidogenic cells.
  • the vector used in the above method comprises a portion of the promoter region of the DAX-1 DNA sequence and a transgene.
  • the transgene is a therapeutic transgene.
  • the invention further provides a method for screening drugs which bind a DAX-1 protein.
  • This method comprises, as a first step, transfecting a cell with (i) a vector capable of expressing at least the ligand binding domain of a DAX-1 gene linked with a DNA binding domain (DBD) sequence, and (ii) a construct capable of expressing a reporter gene and having a response element to which the DBD binds, wherein the construct is part of the vector or is separate therefrom.
  • the transfected cell is cultured under conditions permitting binding of the expressed DBD to the response element.
  • the transfected cell is contacted with a drug to be screened.
  • the presence of a protein encoded by the reporter gene is then detected, the presence of the protein being indicative of the binding of the drug to DAX-1 protein.
  • Figure 1 shows the position of the DAX-1 gene in Xp21.
  • Figure 2a shows the restriction pattern for two patients from apparently independent pedigrees following amplification of a portion of the DAX-1 cDNA, followed by digestion with Rsal.
  • Figure 2b shows the sequence of the DAX-1 DNA from the same two patients.
  • Figure 3a shows deletion of the DAX-1 locus in two brothers with isolated AHC and reduced gene dosage in their mother.
  • Figure 3b shows confirmation of the mother's carrier status by fluorescence in situ hybridization (FISH) of the DAX-1 cosmid.
  • Figure 4 shows the results of a prenatal diagnosis by FISH performed with the DAX-1 cosmid.
  • Figure 4a shows deletion of DAX-1 in the obligate carrier sister of the consultand.
  • Figures 4b and 4c show normal hybridization in cells from the pregnant consultand (4b) and her male fetus (4c).
  • Figure 5 shows the amino acid sequence in the putative zinc finger domain of the DAX-1 protein.
  • Figure 6 shows the locations of the SSCP fragments in the DAX-1 gene.
  • Figure 7a shows the genomic organization of the DAX-1 gene.
  • Figure 7b shows the nucleotide sequence of a portion of exon 1 and the 5 '-flanking region.
  • Figure 8a shows alignment of the C-terminal ligand binding domain of several members of the nuclear hormone receptors, using DAX-1 as the basis for comparison.
  • Figures 8b and 8c show amino acid alignment of the conserved regions II and III of same group of the nuclear hormone receptors.
  • Figure 9 shows polymorphisms and mutations in the DAX-1 gene.
  • Figure 9a shows nineteen DNA samples from different individuals.
  • Figure 9b shows GGAA tetra-nucleotide tandem repeat in an AHC affected family showing Mendelian inheritance of the polymorphism.
  • Figure 9c shows the results of amplication and sequencing of the 266bp fragment of DNA for patient DT.
  • Figure 9d shows the results of amplication and sequencing of the 201bp fragment of DNA for patient JLB.
  • Figure 9e shows the results of amplication and sequencing of the 201bp fragment of DNA for patient PS.
  • Figure 10 is a multiple interspecies zoo blot.
  • Figure 11 is a comparison of zinc coordinating domains of DNA binding proteins.
  • Figure 12 shows the nucleotide and predicted amino-acid sequence of
  • Figure 13 shows a proposed strategy for gene therapy for disorders of adrenocortical development or metabolism.
  • Figure 14 shows relative locations of the genes responsible for cytomegalic adrenal hypoplasia congenita (AHC), glycerol kinase deficiency (GK), and Duchenne's muscular dystrophy (DMD) within the X chromosome.
  • AHC cytomegalic adrenal hypoplasia congenita
  • GK glycerol kinase deficiency
  • DMD Duchenne's muscular dystrophy
  • Figure 15 shows positional cloning of the DAX-1 gene in Xp21.
  • Figure 16 shows results of a Southern blot of a restriction digest from contig cosmids hybridized with the PCR amplified, labeled human fetal adrenal cDNA library.
  • Figure 17 shows the genomic structure of the DAX-1 gene.
  • Figure 18 shows a comparison of the domain structure of DAX-1 to a prototypic member of the nuclear hormone receptor superfamily.
  • Figure 19 shows a comparison of the ligand binding domain of DAX-1 to other members of the nuclear hormone receptor superfamily.
  • Figure 20 shows alignment of the amino acids within regions II and III.
  • Figure 21 shows a comparison of the AF-2 or t4 transactivation domain of DAX-1 to other nuclear hormone receptors.
  • Figure 22a shows alignment of the 3.5 repeats within the amino-terminus of DAX-1.
  • Figure 22b shows a comparison of the putative DAX-1 zinc finger to zinc fingers found in other double stranded DNA binding proteins.
  • Figure 23 is a schematic of the DAX-1 protein illustrating the approximate location of various types of mutations causing AHC. Below the schematic is a Table containing all of the known mutations in the DAX-1 gene.
  • therapeutic gene means DNA encoding an amino acid sequence corresponding to a functional protein or peptide capable of exerting a therapeutic effect on diseased or damaged cells or having a regulatory effect on the expression of a function in diseased or damaged cells.
  • soluble means unbound or circulating DAX-1 molecule such as a portion of DAX-1 fused genetically or chemically to a biologically or chemically active molecule which induces solubility, e.g., DAX-1 with an Ig tail.
  • nucleic acid molecule includes both DNA and RNA and, unless otherwise specified, includes both double-stranded, single-stranded nucleic acids, and nucleosides thereof. Also included are hybrids such as DNA-RNA hybrids, DNA-RNA-protein hybrids, RNA-protein hybrids and DNA-protein hybrids. Reference to a nucleic acid sequence can also include modified bases as long as the modification does not significantly interfere either with binding of a ligand such as a protein by the nucleic acid or Watson-Crick base pairing.
  • enhancer element is a DNA sequence that increases the activity of gene expression promoters for transcription. The enhancer element can be functional upstream, downstream, and in promoters without significant loss of activity.
  • DAX-1 dosage sensitive sex reversal locus, adrenal hypoplasia congenita on chromosome X, gene number 1.
  • operatively linked means to be connected to an expression control sequence in such a way so as to enable the expression of a protein encoded by the gene sequence.
  • recombinant protein means a protein molecule that is made using genetic engineering techniques.
  • recombinant DNA means a DNA molecule that is made using genetic engineering techniques.
  • hybrid means a nucleic acid molecule containing both DNA and RNA, DNA and protein, RNA and protein or DNA and RNA and protein.
  • screening means to select a target drug by testing samples of drugs for their effect on expression of a selected gene.
  • a drug of interest means a drug that is to be tested for its function in activating the expression of a reporter gene.
  • compatible cell means the cell is able to be transformed by a recombinant DNA using techniques in genetic engineering.
  • reporter gene means a gene whose expression can be readily detected in an assay.
  • retinoic acid response element means a nucleic acid sequence that can be bound by a retinoic acid receptor.
  • transgene means a gene whose expression is driven by the promoter element located upstream.
  • DNA binding domain (DBD) sequence means a nucleic acid sequence that encodes a protein domain that is capable of binding a response element on a nucleic acid molecule. Suitable examples include conventional DBDs such as LexA and Gal4. Also included are the DBDs of members of the nuclear hormone receptor superfamily, such as the glucocorticoid receptor and the retinoic acid receptor. Also included are non-conventional DBDs, such as the DBD of DAX-1 or SHP (W Seol et al. (1996) Science 272:1336-1338).
  • response element is a nucleotide sequence which effects an increase or decrease in gene expression when bound by a DNA-binding protein.
  • a response element is responsive to a DBD if the response element effects an increase or decrease in gene expression in response to the binding of the corresponding DNA-binding protein to the response element.
  • adrenocortical metabolism and development includes adrenal hypoplasia congenita (AHC), hypogonadotropic hypogonadism (HH), adrenocortical hyperplasia, adrenal aplasia, congenital adrenal hyperplasia, adrenal leukodystrophy, Addison's disease, adrenocortical adenoma, adrenocortical carcinoma and other disorders affecting or involving the adrenal cortex.
  • AHC adrenal hypoplasia congenita
  • HH hypogonadotropic hypogonadism
  • adrenocortical hyperplasia adrenal aplasia
  • congenital adrenal hyperplasia adrenal leukodystrophy
  • Addison's disease adrenocortical adenoma
  • adrenocortical carcinoma affecting or involving the adrenal cortex.
  • DAX-1 Protein Molecules
  • the invention provides an isolated protein designated DAX-1 which binds a retinoic acid response element.
  • the protein can be a human protein, a monkey protein, a mouse protein, a
  • the protein has the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in Figure 12.
  • the protein is a recombinant protein having an amino acid sequence substantially as shown in Figure 12.
  • the protein is a heterodimeric protein comprising
  • DAX-1 and a nuclear hormone receptor Suitable examples of nuclear hormone receptors include, but are not limited to, SF-1, thyroid hormone receptor, androgen receptor, estrogen receptor, progesterone receptor, glucocorticoid receptor, mineralocorticoid receptor, retinoic acid receptor, retinoid X receptor and COUP.
  • the heterodimeric protein can comprise DAX-1 and SFI, or DAX-1 with any other nuclear hormone receptor.
  • DAX-1 protein molecules may be embodied in many forms.
  • Embodiments of the DAX-1 protein include a purified DAX-1 protein and a functional, soluble DAX-1 protein.
  • the purified DAX-1 protein molecule is substantially free of other proteins or molecules which impair the binding of DAX-1 to a retinoic acid or other response element.
  • a functional soluble DAX-1 protein has the amino acid sequence beginning with methionine at position 1 and ending with isoleucine at position 470 as shown in Figure 12 or a fragment thereof.
  • soluble means unbound or circulating DAX-1 such as a portion of DAX-1 fused (genetically or chemically) to a biologically or chemically active molecule which induces solubility, e.g., DAX-1 with an Ig tail (DAX-l/Ig).
  • DAX-1 with an Ig tail
  • the functional, soluble DAX-1 protein or fragment thereof retains its ability to bind the retinoic acid or other response element.
  • DAX-1 protein molecules of the invention may have amino acid changes (e.g., substitutions or deletions of one or more amino acid residues) within the molecule depending on the species expressing the DAX-1 protein (for example, the human DAX-1 protein shares about 65% identity and 75% similarity with the mouse DAX-1 protein) and the protein molecule will retain DAX-1 activity.
  • Amino acid substitutions include, but are not necessarily limited to, amino acid substimtions known in the art as "conservative".
  • glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V).
  • Methionine (M) which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine.
  • Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered "conservative" in particular environments.
  • the invention provides a nucleic acid molecule encoding a DAX-1 protein.
  • the nucleic acid encodes a recombinant DAX-1 protein; in another embodiment, the nucleic acid molecule has a nucleotide sequence as shown in Figure 12.
  • the invention further provides a recombinant DNA molecule comprising a nucleotide sequence encoding a DAX-1 protein operatively linked to an expression control sequence.
  • the invention also provides nucleic acid molecules encoding the protein of the invention ( Figure 12).
  • the nucleic acid molecule is a DNA molecule.
  • the genomic DNA molecule includes both the intron and exons of the sequence which encodes DAX-1.
  • the nucleic acid molecule is a cDNA molecule as shown in Figure 12.
  • the nucleic acid molecule is a ribonucleic acid (RNA).
  • the nucleic acid molecule is a hybrid DNA/RNA, DNA/protein, RNA/protein or DNA/RN A/protein molecule.
  • the invention also encompasses nucleic acid molecules that hybridize to the above-mentioned nucleic acid molecules of the invention and encode a protein having DAX-1 activity.
  • the hybridization is under conventional hybridization conditions, preferably under stringent conditions.
  • the invention also encompasses DNA that encode anti-sense RNAs or specific ribozymes which allow the control of the expression of the nucleic acid molecules of the invention in desired host and target cells.
  • the anti-sense RNAs and specific ribozymes are also encompassed by the invention.
  • the invention relates to primers which allow the specific amplification of nucleic acid molecules of the invention or of any specific parts thereof.
  • the invention relates to probes that specifically hybridize to nucleic acid molecules of the invention or to any part thereof.
  • the invention further provides an expression vector comprising a recombinant DNA molecule encoding a DAX-1 protein.
  • the invention further provides a vector having the nucleic acid molecule discussed hereinabove and a transgene.
  • the expression vector comprises a DNA molecule encoding a DAX-1 protein and a transgene.
  • the vector can comprise a portion of the DAX-1 promoter.
  • the portion of the DAX-1 promoter is the SF-1 response element located upstream of the TATA box in the 5' flanking region of the DAX-1 gene.
  • the vector can further comprise a suitable enhancer element.
  • suitable enhancer elements include but are not limited to a portion of the SF-1 promoter, a portion of the CMV promoter, a poition of the MMLV promoter, a poition of the SV40 promoter, a portion of the RSV promoter, or the SF-1 response element located upstream of the TATA box in the 5' flanking region of the DAX-1 gene.
  • the transgene is a therapeutic gene.
  • the therapeutic gene may encode a cytokine, a tumor suppressor, a growth factor, cytotoxin, or an antisense RNA molecule.
  • the therapeutic gene may be an oncogene.
  • the therapeutic gene may serve to provide or augment expression of a deficient product to compensate for the deficiency, or the therapeutic gene may serve to inhibit the growth of or kill diseased cells associated with adrenocortical disorders of metabolism and development, or to produce cytokines or other cytotoxic agents which directly or indirectly inhibit the growth of or kill such diseased cells.
  • Examples of therapeutic genes which compensate for a deficiency include a gene encoding 21 -hydroxylase, which can be used to compensate for a deficiency in 21 -hydroxylase by increasing the expression of this enzyme in adrenal cells.
  • Adrenal disorders, including 21 -hydroxylase deficiency, are discussed in CJ
  • therapeutic genes include suicide genes. These are gene sequences the expression of which produces a protein or agent that inhibits mmor cell growth or induces cell death (CA. Mullen, Pharmac. Ther. Vol. 63, pp. 199- 207, 1994).
  • Suicide genes include genes encoding enzymes, oncogenes, tumor suppressor genes, genes encoding toxins, genes encoding cytokines, or a gene encoding oncostatin.
  • the purpose of the therapeutic gene is to inhibit the growth of or kill diseased cells associated with adrenocortical disorders of metabolism and development or produce cytokines or other cytotoxic agents which directly or indirectly inhibit the growth of or kill such diseased cells.
  • suicide genes include, but are not limited to, genes which can induce programmed cell death such as the reaper gene.
  • Suitable enzymes include thymidine kinase (TK), xanthine-guanine phosphoribosyltransferase (GPT) gene from E. Coli or E. Coli cytosine deaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT).
  • TK thymidine kinase
  • GPT xanthine-guanine phosphoribosyltransferase
  • CD E. Coli or E. Coli cytosine deaminase
  • HPRT hypoxanthine phosphoribosyl transferase
  • Treatment with a therapeutic transgene encoding an enzyme can render the transfected cells sensitive to subsequent drug treatment.
  • adrenal cells are transfected with the TK gene and thereby become senstive to ganciclovir or acyclo
  • Suitable oncogenes and mmor suppressor genes include neu, EGF, ras (including H, K, and N ras), p53, Retinoblastoma mmor suppressor gene (Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase (PTPase), and nm23.
  • Suitable toxins include Pseudomonas exotoxin A and S; diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-like toxins (SLT-1, -2), ricin, abrin, supporin, and gelonin.
  • Suitable cytokines include interferons, GM-CSF interleukins, mmor necrosis factor (TNF) (Wong G, et al., Human GM-CSF: Molecular cloning of the complementary DNA and purification of the namral and recombinant proteins. Science 1985; 228:810; WO9323034 (1993); Horisberger MA, et al., Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional smdy of the corresponding gene promoter. Journal of Virology.
  • Growth factors include Transforming Growth Factor- ⁇ (TGF ⁇ ) and ⁇ (TGF ⁇ ), cytokine colony stimulating factors (Shimane M, et al., Molecular cloning and characterization of G-CSF induced gene cDNA. Biochemical and Biophysical Research Communications. 1994 Feb 28, 199(l):26-32; Kay AB, et al., Messenger RNA expression of the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, and granulocyte/macrophage colony-stimulating factor, in allergen-induced late-phase cutaneous reactions in atopic subjects. Joumal of Experimental Medicine, 1991 Mar 1, 173(3):775-8; de Wit H, et al., Differential regulation of M-CSF and IL-6 gene expression in monocytic cells. British Journal of Haematologv. 1994 Feb,
  • Cytokines include but are not limited to interferons, interleukins and colony stimulating factors.
  • Colony stimulating factors include but are not limited to granulocyte colony stimulating factor, or granulocyte macrophage colony stimulating factor.
  • the invention provides a host vector system comprising an expression vector encoding a DAX-1 protein, transfected into a compatible cell.
  • the compatible cell is a bacterial cell or a eucaryotic cell.
  • An example of a compatible eucaryotic host cell is an adrenal cell.
  • the vector can be a plasmid, cosmid or phage vector encoding the cDNA molecule discussed above.
  • the invention provides a host-vector system comprising the plasmid, cosmid or phage vector transfected into a compatible eucaryotic host cell.
  • compatible eucaryotic host cells include a yeast cell, a plant cell, or an animal cell.
  • the host- vector system is useful for the production of a DAX-1 protein.
  • the invention provides a method of producing a DAX-1 protein.
  • the method comprises growing a host vector system so as to produce the DAX-1 protein and recovering the DAX-1 protein so produced.
  • the invention further provides a method of producing a DAX-1 protein in vitro.
  • This methods comprises transcribing a vector comprising a nucleic acid molecule encoding a DAX-1 protein in the presence of transcribable RNA, thereby creating mRNA. Additionally, the method requires translating the mRNA transcribed in the above step so as to provide DAX-1 protein. As a fmal step, the DAX-1 protein is recovered.
  • the invention further provides a method for inhibiting the proliferation of diseased cells associated with the presence of a DAX-1 protein.
  • a DAX-1 protein an adrenal or other steroidogenic cell is transfected with the recombinant DNA of the invention under conditions that the DAX-1 protein is expressed.
  • the expression of the DAX-1 protein results in the inhibition of the proliferation of diseased cells.
  • the proliferation of adrenal cells is inhibited by increased expression of DAX-1 protein.
  • the invention further provides a method of transferring a therapeutic gene into steroidogenic cells of the hypothalamic-pituitary-adrenal/gonadal axis.
  • This method comprises introducing a vector comprising a DAX-1 expression control sequence operatively linked to a therapeutic gene into a producer cell which results in the integration of the vector into the producer cell.
  • the vector is integrated into the genome of the producer cell.
  • the vector is not integrated into the genome of the producer cell.
  • this method requires selecting a producer cell having the vector integrated into the cell.
  • the producer cell so selected is grafted proximate to the steroidogenic cells, so that the steroidogenic cells are infected with the vector produced by the producer cell and the therapeutic gene is transferred to the steroidogenic cells.
  • This invention also involves targeting a gene-of-interest to diseased cells associated with adrenocortical disorders of metabolism and development so that the protein encoded by the gene is expressed and directly or indirectly ameliorates the diseased state. Targeting strategies are known in the art and are reviewed in A.L. Joyner, ed., Gene Targeting: A Practical Approach, Oxford University Press: New York, 1993.
  • the transgene driven by a specific promoter in the vector expresses the protein encoded by the gene.
  • the use of the highly specific DAX-1 gene vector allows selective expression of the specific genes in steroidogenic cells.
  • the present invention relates to a process for administering modified vectors into steroidogenic cells, for example, of the adrenal gland. More particularly, the invention relates to the use of vectors carrying functional therapeutic genes to produce molecules that are capable of directly or indirectly affecting cells in the adrenal gland to repair damage sustained by the cells from defects, disease or trauma.
  • vectors of the invention include a therapeutic gene or transgenes.
  • the genetically modified vectors are administered into the subject to treat defects, disease such as AHC or HH by introducing a therapeutic gene product or products into the subject that enhance the production of endogenous molecules that have ameliorative effects in vivo.
  • the basic tasks in the present method of the invention are isolating the gene of interest, selecting the proper vector vehicle to deliver the gene of interest to the body, administering the vector having the gene of interest into the body, and achieving appropriate expression of the gene of interest.
  • the present invention provides packaging the cloned genes, i.e. the genes of interest, in such a way that they can be injected directly into the bloodstream or into relevant organs of patients who need them.
  • the packaging will protect the foreign DNA from elimination by the immune system and direct it to appropriate tissues or cells. Examples of packaging include, but are not limited to, producer cells, liposomes and retroviruses. Packaging cells are described in PM Patel et al. (1993) in: G Gallagher et al., eds. Tumour Immunobiology: A Practical Approach, New York:Oxford, 296-303. Liposome packaging is described in MJ Stewart et al., Hum. Gene Ther. (1992) 3:267-275.
  • Universal donor cells can be used to deliver the gene of interest; the donor cells may be heterologous, homologous or autologous. Immune responses to the donor material can be reduced by treatment with immunosuppression agents or by shielding the donor material with immunoisolation devices, such as the packaging discussed above.
  • another gene e.g., a selectable marker, can be inserted that will allow easy identification of cells that have incorporated the modified retrovirus.
  • the critical focus on the process of gene therapy is that the new gene must be expressed in target cells at an appropriate level with a satisfactory duration of expression.
  • HSV-TK Herpes Simplex Virus Thymidine Kinase
  • Vectors for use in the gene therapy methods of the present invention include, but are not limited to, viral vectors such as adenoviruses, retroviral vectors, or adeno-associated viral (AAV) vectors.
  • viral vectors such as adenoviruses, retroviral vectors, or adeno-associated viral (AAV) vectors.
  • AAV adeno-associated viral
  • the viral vector selected should meet the following criteria: 1) the vector must be able to infect the desired cells and thus viral vectors having an appropriate host range must be selected; 2) the transferred gene should be capable of persisting and being expressed in a cell for a suitable period of time; and 3) the vector should be safe to the host.
  • Retroviral vectors and adenoviruses offer an efficient, useful, and presently the best-characterized means of introducing and expressing foreign genes efficiently in mammalian cells. These vectors have very broad host and cell type ranges, express genes stably and efficiently. The safety of these vectors has been proved by many research groups. In fact many are in clinical trials.
  • retroviruses such as Moloney murine leukemia virus (MMLV); papovaviruses such as JC, SV40, polyoma, adenoviruses; Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovine papilloma virus type I (BPV); vaccinia and poliovirus and other human and animal viruses.
  • Adenoviruses have several properties that make them attractive as cloning vehicles (Bachettis et al. : Transfer of gene for thymidine kinase-deficient human cells by purified herpes simplex viral DNA. PNAS USA. 1977 24:1590; Berkner, K.L. : Development of adenovirus vectors for expression of heterologous genes. Biotechniques, 1988 6:616; Ghosh-Choudhury G, et al., Human adenovirus cloning vectors based on infectious bacterial plasmids.
  • adenoviruses possess an intermediate sized genome that replicates in cellular nuclei; many serotypes are clinically innocuous; adenovirus genomes appear to be stable despite insertion of foreign genes; foreign genes appear to be maintained without loss or rearrangement; and adenoviruses can be used as high level transient expression vectors with an expression period up to 4 weeks to several months. Extensive biochemical and genetic smdies suggest that it is possible to substitute up to 7-7.5 kb of heterologous sequences for native adenovirus sequences generating viable, conditional, helper-independent vectors (Kaufman
  • AAV is a small human parvovirus with a single stranded DNA genome of approximately 5 kb. This virus can be propagated as an integrated provirus in several human cell types.
  • AAV vectors have several advantages for human gene therapy. For example, they are trophic for human cells but can also infect other mammalian cells; (2) no disease has been associated with AAV in humans or other animals; (3) integrated AAV genomes appear stable in their host cells; (4) there is no evidence that integration of AAV alters expression of host genes or promoters or promotes their rearrangement; (5) introduced genes can be rescued from the host cell by infection with a helper virus such as adenovirus.
  • helper virus such as adenovirus
  • HSV-1 vector system facilitates introduction of virtually any gene into non ⁇ mitotic cells (Geller et al. an efficient deletion mutant packaging system for a defective herpes simplex virus vectors: Potential applications to human gene therapy and neuronal physiology. PNAS USA. 1990 £2:8950).
  • bovine papilloma virus- based vector (Sarver N, et al., Bovine papilloma virus DNA: A novel eukaryotic cloning vector. Mol Cell Biol 1981; 1:486).
  • Vaccinia and other poxvirus-based vectors provide a mammalian gene transfer system.
  • Vaccinia virus is a large double-stranded DNA virus of 120 kilodaltons (kd) genomic size (Panicali D, et al., Construction of poxvirus as cloning vectors: Insertion of the thymidine kinase gene from herpes simplex virus into the DNA of infectious vaccine virus. Proc Natl Acad Sci USA 1982; 79:4927; Smith et al. infectious vaccinia virus recombinants that express hepatitis B virus surface antigens. Namre. 1983 3_0_2:490.)
  • Retroviruses are packages designed to insert viral genes into host cells (Guild B, et al., Development of retrovirus vectors useful for expressing genes in cultured murine embryonic cells and hematopoietic cells in vivo. J Virol 1988; 62:795; Hock RA, et al., Retrovirus mediated transfer and expression of drug resistance genes in human hemopoietic progenitor cells. Namre 1986; 320:275).
  • the basic retrovirus consists of two identical strands of RNA packaged in a proviral protein.
  • the core is surrounded by a protective coat called the envelope, which is derived from the membrane of the previous host but modified with glycoproteins contributed by the virus.
  • the methods described below to modify vectors and administering such modified vectors into the subject are merely for purposes of illustration and are typical of those that might be used. However, other procedures may also be employed, as is understood in the art.
  • the invention further provides a method for screening drugs which bind a DAX-1 protein.
  • This method comprises, as a first step, transfecting a cell with (i) a vector capable of expressing at least the ligand binding domain of a DAX-1 gene linked with a DNA binding domain (DBD) sequence, and (ii) a construct capable of expressing a reporter gene and having a response element to which the DBD binds.
  • the construct may be part of the vector or separate therefrom.
  • the transfected cell is cultured under conditions permitting binding of the expressed DBD to the response element.
  • the transfected cell is contacted with a drug to be screened.
  • the presence of a protein encoded by the reporter gene is then detected, the presence of the protein being indicative of the binding of the drug to DAX-1 protein.
  • the ligand binding domain (LBD) of the DAX-1 gene as used in the above method includes the E region of the DAX-1 gene, which corresponds to approximately positions 260 to 470 of the nucleotide sequence shown in Figure 12. Also included are conservative variations of the E region, such as trimming the tail of the E region as well as other modifications of the nucleic acid sequence that do not prevent the encoded protein to bind ligand.
  • the LBD of the DAX-1 gene may be linked with a DBD either by fusion with a homologous or heterologous DBD or by inclusion of the DAX-1 DBD in a contiguous sequence.
  • the DAX-1 sequence used in this method may be all or a portion of the nucleotide sequence shown in Figure 12, a nucleotide sequence encoding the amino acid sequence shown in Figure 12, or a nucleotide sequence encoding substantially the same amino acid sequence as that shown in Figure 12.
  • the DBD sequence for use in the above method may be any DBD capable of binding to a response element.
  • DBDs include, but are not limited to, the DBDs from the yeast transcription factors such as Gal4 or LexA, or from nuclear hormone receptors such as DAX-1, SF-1, glucocorticoid receptor or retinoic acid receptor.
  • the response element for use in the above method may be any response element, so long as it is responsive to the DBD.
  • ligand-bound nuclear receptor hormones act in dimeric form to activate expression of a target gene via binding to an appropriate response element.
  • Some members of the nuclear receptor hormone superfamily, such as SHP, are believed to reduce gene expression, possibly by dimerizing with another nuclear receptor hormone and thereby preventing the other receptor from binding the appropriate response element.
  • binding of a drug to a DAX-1 protein will effect an increase in expression of the reporter gene.
  • binding of a drug to a DAX-1 protein would result in a decrease in expression of the reporter gene.
  • Suitable reporter genes for use in the above method include, but are not limited to, luciferase, ⁇ -galactosidase, chloramphenicol acetyl transferase (CAT) and secreted alkaline phosphatase (SeAP). SeAP is secreted directly into the culmre medium, making the assay for reporter gene expression faster and simpler than would be the case for other reporter gene products.
  • luciferase ⁇ -galactosidase
  • CAT chloramphenicol acetyl transferase
  • SeAP secreted alkaline phosphatase
  • the invention further provides antibodies that specifically bind to a DAX-1 protein.
  • the antibodies may be polyclonal or monoclonal.
  • the invention also encompasses antibody fragments which specifically recognize a DAX-1 protein.
  • an antibody fragment is defmed as at least a portion of the variable region of the immunoglobulin molecule which binds to its target, i.e. the antigen binding region. Some of the constant region of the immunoglobulin may be included.
  • the monoclonal antibody of the invention can be produced using well- established hybridoma techniques first introduced by Kohler and Milstein [see, Kohler and Milstein, "Continuous Cultures Of Fused Cells Secreting Antibody Of Pre-Defmed Specificity", Mature, 256:495-97 (1975).
  • lymphocytes are obtained from the animal either from the spleen, lymph nodes or peripheral blood.
  • lymphocytes are obtained from the spleen.
  • the splenic lymphocytes are then fused with a myeloma cell line, usually in the presence of a fusing agent such as polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • myeloma cell lines may be used as a fusion partner according to standard techniques; for example, the P3-NSl/lAg4-l, P3-x63-Ag8.653 or Sp2/O Agl4 myeloma lines. These myeloma lines are available from the American Type Culmre Collection ("ATCC”) in Rockville, Maryland.
  • ATCC American Type Culmre Collection
  • the resulting cells which include the desired hybridomas, are then grown in a selective medium, such as HAT medium, in which unfused parental myeloma or lymphocyte cells evenmally die. Only the hybridoma cells survive and can be grown under limiting conditions to obtain isolated clones.
  • the supematants of the hybridomas are screened for the presence of antibody of that desired specificity, e.g., by immunoassay techniques using the antigen that had been used for immunization. Positive clones can then be subcloned under limiting dilution conditions and the monoclonal antibody produced can be isolated.
  • Hybridomas produced according to these methods can be propagated in vitro or in vivo (in ascites fluid) using techniques known in the art [see, generally, Fink et al., supra at page 123, Fig. 6-11].
  • Commonly used methods for purifying monoclonal antibodies include ammonium sulfate precipitation, ion exchange chromatography, and affinity chromatography [see, e.g., Zola et al., "Techmques For The Production And Characterization Of Monoclonal Hybridoma Antibodies", in Monoclonal Hvbridoma Antibodies: Techniques And Applications. Hurell (ed.), pp. 51-52 (CRC Press 1982)].
  • the invention provides methods of diagnosing adrenocortical disorders such as AHC and HH, which arise from mutations of the DAX-1 gene.
  • the diagnostic methods can be performed on patient samples containing DNA, including but not limited to DNA from white blood cells, DNA from whole blood or DNA from amniocytes.
  • a probe specific for the DAX-1 gene can be detectably labeled and allowed to hybridize in sim with the X chromosomes in the sample.
  • detectable labels for use in this method include fluorescent labels.
  • probes specific for DAX-1 include cosmid 8E10.
  • FISH fluorescent in situ hybridization
  • portions of the nucleic acid sequence shown in Figure 12 and in Figure 7B can be used in conventional nucleic acid amplification protocols, including PCR.
  • Amplified strands can be sequenced accordmg to standard techniques, including the use of commercially available kits (Pharmacia), and/or digested with restriction enzymes (e.g., Rsa I).
  • Digested fragments may then be analyzed by, for example, dot hybridization and or gel electrophoresis, and compared to digested fragments prepared from normal subjects, with alterations in level of hybridization or fragment size being indicative of an abnormality in the DAX-1 gene.
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid molecule of the invention or an expression vector encoding a DAX-1 protein or encoding a fragment thereof and, optionally, a suitable carrier.
  • a suitable carrier includes any material which when combined with the nucleic acid or other molecule of the invention retains the molecule's activity and is non-reactive with the subject's immune systems.
  • suitable carrier includes, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Other carriers may also include sterile solutions, tablets including coated tablets and capsules.
  • Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients.
  • Compositions comprising such carriers are formulated by well known conventional methods.
  • the invention also provides a diagnostic composition comprising a nucleic acid molecule of the invention, a probe that specifically hybridizes to a nucleic acid molecule of the invention or to any part thereof, or an antibody or fragment thereof of the invention.
  • Suitable vectors contaimng the desired therapeutic gene coding and control sequences employs standard ligation and restriction techniques, which are well understood in the art (see Maniatis et al., in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences, or synthesized oligonucleotides are cleaved, tailored, and religated in the form desired.
  • Site-specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by the manufacmrer of these commercially available restriction enzymes (See, e.g. New England Biolabs Product Catalog).
  • suitable restriction enzyme or enzymes
  • about 1 mg of plasmid or DNA sequences is cleaved by one unit of enzyme in about 20 ⁇ l of buffer solution.
  • an excess of restriction enzyme is used to insure complete digestion of the DNA substrate.
  • Restriction cleaved fragments may be blunt ended by treating with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using incubation times of about 15 to 25 min at 20°C to 25°C in an appropriate buffer (e.g., 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 ⁇ M dNTPs).
  • an appropriate buffer e.g., 50 mM Tris (pH 7.6) 50 mM NaCl, 6 mM MgCl 2 , 6 mM DTT and 5-10 ⁇ M dNTPs.
  • the Klenow fragment fills in at 5' sticky ends but chews back protruding 3' single strands, even though the four dNTPs are present.
  • selective repair can be performed by supplying only one of the dNTPs, or with selected dNTPs, within the limitations dictated by the namre of the sticky ends.
  • the mixmre is extracted with phenol/chloroform and ethanol precipitated.
  • Treatment under appropriate conditions with a nuclease results in hydrolysis of any single-stranded portion.
  • Ligations are performed in 10-50 ⁇ l volumes under the following standard conditions and temperatures using T4 DNA ligase. Ligation protocols are standard (D. Goeddel (ed.) Gene Expression Technology: Methods in Enzymology (1991)).
  • vector fragments In vector construction employing "vector fragments", the vector fragment is commonly treated with bacterial alkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP) in order to remove the 5' phosphate and prevent religation of the vector. Alternatively, religation can be prevented in vectors which have been double digested by additional restriction enzyme digestion of the unwanted fragments.
  • Suitable vectors include viral vector systems e.g. ADV, RV, and AAV (R.J.
  • non-vector methods include nonviral physical transfection of DNA into cells; for example, microinjection (DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediated transfection (Feigner et al., Proc. Natl. Acad. Sci. USA. 84:7413-7417 (1987), Feigner and Holm, Focus 11:21-25 (1989) and Feigner et al., Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods known in the art.
  • a vesicle can be constructed in such a way that its membrane will fuse with the outer membrane of a target cell.
  • the vector of the invention in vesicles can home into the adrenal cells.
  • the spheroplasts are maintained in high ionic strength buffer until they can be fused through the mammalian target cell using fusogens such as polyethylene glycol.
  • Liposomes are artificial phospholipid vesicles. Vesicles range in size from 0.2 to 4.0 micrometers and can entrap 10% to 40% of an aqueous buffer containing macromolecules. The liposomes protect the DNA from nucleases and facilitate its introduction into target cells. Transfection can also occur through electroporation.
  • the modified vectors are suspended in complete PBS at a selected density for injection.
  • any osmotically balanced solution which is physiologically compatible with the subject may be used to suspend and inject the modified vectors into the host.
  • the cell suspension is drawn up into the syringe and administered to anesthetized recipients. Multiple injections may be made using this procedure.
  • the viral suspension procedure thus permits administration of genetically modified vectors to any predetermined site in the subject, is relatively non-traumatic, allows multiple administrations simultaneously in several different sites or the same site using the same viral suspension. Multiple injections may consist of a mixmre of therapeutic genes.
  • Transcription initiation is an early and critical event in gene expression. This depends on the promoter and enhancer sequences and is influenced by specific cellular factors that interact with these sequences.
  • the transcriptional unit of many prokaryotic genes consists of the promoter and in some cases enhancer or regulatory elements (Banerji et al., Cell 27:299 (1981); Corden et al., Science 209:1406 (1980); and Breathnach and Chambon, Ann. Rev. Biochem 50:349 (1981)).
  • LTR long terminal repeat
  • Moloney murine leukemia virus (MMLV) and Rous sarcoma virus (RSV) LTRs contain promoter and enhancer sequences (Jolly et al., Nucleic Acids Res. 11:1855 (1983); Capecchi et al., In: Enhancer and eukaryotic gene expression, Gulzman and Shenk, eds., pp. 101-102, Cold Spring Harbor Laboratories, Cold Spring Harbor, New York).
  • the present invention provides methods for maintaining and increasing expression of therapeutic genes using a DAX-1 adrenal cell specific promoter.
  • an enhancer sequence may be used to increase the level of therapeutic gene expression. Enhancers can increase the transcriptional activity not only of their native gene but also of some foreign genes (Armelor, Proc. Natl. Acad. Sci. USA 70:2702 (1973)).
  • CMV enhancer sequences can be used with the DAX-1 promoter to increase therapeutic gene expression.
  • Therapeutic gene expression may also be increased for long term stable expression after injection using cytokines to modulate promoter activity.
  • modified vectors carrying a therapeutic gene are injected systemically or into the vicinity of the adrenal gland of a subject.
  • a protein product can be expressed comprising growing the host vector system of the invention so as to produce the protein in the host and recovering the protein so produced.
  • This method permits the expression of genes of interest in both unicellular and multicellular organisms.
  • adrenal cells having the vector of the invention comprising a gene of interest e.g., the ras gene
  • a gene of interest may be used in microtiter wells as an unlimited source for the ras gene product.
  • a sample from a subject would be added to the wells to detect the presence of antibodies directed against the ras gene.
  • This assay can aid in the quantitative and qualitative determination of the presence of ras antibodies in the sample for the clinical assessment of whether the subject's immune system is combatting the disease associated with elevated levels of ras.
  • AHC or HH can be treated via gene therapy, i.e., the correction of a disease phenotype in vivo through the use of the nucleic acid molecules of the invention.
  • the subject of the gene therapy may be a human, equine, porcine, bovine, murine, canine, feline, or avian subject.
  • Other warm blooded animals are also included in this invention.
  • Figure 13 shows a strategy for gene therapy for disorders of adrenocortical development or metabolism.
  • a promoter that would target expression to the adrenal cortex such as the DAX-1 promoter, would be used with the coding sequence for the gene specific for the patient's disorder.
  • the vector containing this construct would be introduced systemically, but expression would be targeted to the adrenal cortex.
  • other administration means are possible.
  • the most effective mode of administration and dosage regimen for the molecules of the present invention depends upon the exact location of the diseased cells being treated, the severity and course of the disease, the subject's age, health and response to treatment and the judgment of the treating physician. Accordingly, the dosages of the molecules should be titrated to the individual subject.
  • the molecules may be delivered directly or indirectly via another cell, autologous cells are preferred, but homologous and heterologous cells are encompassed within the scope of the invention.
  • the interrelationship of dosages for animals of various sizes and species and humans based on mg/m 2 of surface area is described by Freireich, E.J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966). Adjustments in the dosage regimen may be made to optimize the disease cell growth inhibiting and killing response, e.g., doses may be divided and administered on a daily basis or the dose reduced proportionally depending upon the simation (e.g., several divided doses may be administered daily or proportionally reduced depending on the specific therapeutic simation).
  • ADVANTAGES OF THE INVENTION Molecular genetic screening of heritable disease syndromes is a rapidly evolving field of molecular medicine which will have a significant impact on human health in this and the next century.
  • the identification of mutations associated with a vast number of heritable disease syndromes has considerable therapeutic value for affected individuals and allows more accurate genetic counseling for affected families.
  • DAX-1 X-linked adrenal hypoplasia congenita
  • HH hypogonadotropic hypogonadism
  • Applicant's discovery allows for the screening and diagnosis of the disease syndromes associated with the DAX-1 gene. Specifically, this invention allows for a quick and efficient analysis of the nucleic acid sequences which are affected in AHC and HH. In addition, the identification of the DAX-1 protein coding region allows for the generation of reagents such as monoclonal antibodies which have further diagnostic potential. This invention is illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow.
  • the DAX-1 cDNA was cloned by the method of cDNA amplification for identification of genomic expressed sequences as summarized below.
  • JK2 genomic DNA was used to screen the a human X chromosome cosmid library (LLOXNCOl "U", Lawrence Livermore National Laboratory, Livermore, CA) and identified two positive clones. The ends of these two cosmids were used as starting points to produce a complete cosmid contig between the markers YHX133R and JK2 ( Figure 1). The ends of the cosmids were hybridized against the library described above, in order to walk in the telomeric and centromeric directions. The additional markers, QST59, 332F10R, A107E5R, YHX133L and K23-b2, were used to characterize the 15 cosmids in the contig.
  • the cosmids in the contig were digested with EcoRI, Southern blotted and hybridized with a human fetal adrenal cDNA library in lambda gtll (HLlll ⁇ b Clontech, Palo Alto, CA) that had been amplified with primers to the vector ends and labeled as previously described.
  • Cosmid 8E10 which was used for FISH analysis (see below), contained both exons of DAX-1 and the approximately 3-kb intron ( Figure 1).
  • Cosmid 8E10 mapped between A107E5R and YHX133L, and included the three EcoRI fragments contaimng the DAX-1 gene, ordered 3.4-kb, 1.4-kb and 4.0-kb, centromeric to telomeric, the direction of transcription. Exon 1 was contained in the 3.4-kb fragment and Exon 2 was contained in the 4.0-kb fragment.
  • Figure 1 shows the position of the DAX-1 gene in Xp21.
  • the figure is oriented with the telomere (Xp-ter) to the left and the centromere (Xp-cen) to the right.
  • Yeast artificial chromosomes that have been previously mapped are shown as horizontal bars along the top, and markers in this region are shown at their approximate positions on the chromosome (hatched horizontal bar).
  • the cosmid contig is shown below the chromosome with cosmid 8E10 indicated and vertical bars representing the DAX-1 exons.
  • the region containing DAX-1 is shown at higher resolution below the cosmids, with its two exons and single intron, and the EcoRI restriction sites.
  • the DAX-1 gene is transcribed in the direction from centromere to telomere (right to left).
  • DNAs that were successfully amplified by all primer pairs were assumed to have a point mutation or small deletion or insertion, and the amplified products were digested with a series of restriction enzymes and subjected to automated sequencing. If amplification of a patient's DNA was unsuccessful with the DAX-1 primers, then the cell line from that patient was examined by FISH with cosmid
  • TP is a 6 y.o. male who was bom at 42 weeks gestation to a G4 P3 Abl (SAB at 8 wks) LC2 (two female sibs) mother, who had a normal pregnancy, labor and delivery. His birth weight was 81b. 3oz. and nursery discharge weight was 81b. 6oz. At 3 weeks of age he was hospitalized because of failure to thrive (weight 81b. 3oz.) and had a poor suck, fatigue, hyponatremia (Na+ 117 meq/L) and hyperkalemia (K+ 6.4 meq/L). He was started on glucocorticoid and mineralocorticoid replacement therapy and was treated with supplemental NaCl. In a later evaluation with an ACTH stimulation test (0.15 mg Cortrisin in 250 ml normal saline), his cortisol levels were unresponsive at 3 mmol/L.
  • ACTH stimulation test (0.15 mg Cortrisin in 250 ml normal
  • His plasma ACTH rose from 249 pg/ml to 1490 pg/ml after withdrawal of his replacement steroids. He had an LRF stimulation test that showed maximum changes in his LH of 12.7 IU/L and FSH of 1.3 IU/L. The interpretation of these data was that he had an unresponsive adrenal gland, but a normal gonadotropic axis for his age. Therefore, at 17 months of age he was diagnosed with isolated AHC; his developmental evaluation was normal.
  • LC He was unresponsive after ACTH stimulation (15 units every 12 hrs intramuscularly for 3 days) and he was diagnosed with Addison disease. The uncle had delayed puberty and was treated with testosterone at 20 years of age. LC, 11 years of age, and NC, 8 1/2 years of age, are full male siblings. LC first came to medical attention because of recurrent vomiting, failure to thrive in infancy and hyperpigmentation. Diagnostic testing suggested AHC, and he was placed on glucocorticoids and mineralocorticoids at approximately 6 months of age. His younger brother, NC, developed adrenal crisis shortly after birth, and was started on glucocorticoid and mineralocorticoid replacement therapy at 2 weeks of age. Over the next several years, NC developed numerous episodes of adrenal crisis requiring hospitalization, whereas, LC has never had episodes of adrenal crisis.
  • NC had cryptorchidism, necessitating orchiopexy at 4 years of age and both boys have mild to moderate developmental disabilities.
  • Family history is negative for other individuals affected with disorders of adrenal function.
  • OM the consultand in the fourth family, and VB are adult sisters, and FMB and DMB are the sons of VB.
  • OM a 36 y.o. female, was imtially seen for prenatal genetic counseling because of advanced maternal age. The family history indicated numerous males dying in the neonatal period in a pattem consistent with X-linked inheritance.
  • FMB the first son of VB died at 3 weeks of age after becoming weak and lethargic.
  • the carrier mother of RE showed the 205-bp, 108-bp and 98-bp bands ( Figure 2).
  • the RE and TP cell lines they were characterized by two different polymorphic markers, DXS52 and the 3' end of the apoB gene. Different polymorphic patterns were observed with each cell line and with both markers, showing that the two cell lines were distinct.
  • Figure 3a shows previously mapped PCR markers, ordered from telomere (top) to centromere (bottom). These additional markers indicated that the telomeric breakpoint was between A227C1R, DAX-1 (BF-BR) and the centromeric breakpoint was between K23-b2 and K23-6P (Figure 3).
  • the affected boys were deleted for DAX-1 (BF BR) and K23-b2, and the amplifications of these markers in their mother appeared to be diminished compared to the normal control, suggesting a deletion in one of her X chromosomes.
  • Analysis of PCR amplification products in the mother suggested the presence of reduced gene dosage (Figure 3), consistent with carrier stams, although the difference was not conclusive.
  • the mutation in this family was established by analysis of cells from VB, who was the sister of the consultand, OM, and an obligate heterozygote by pedigree analysis.
  • FISH analysis of whole blood from VB with a cosmid containing the DAX-1 gene, cosmid 8E10, and an X centromere control probe, PDMX-1 showed deletion of the DAX-1 locus from one of her X chromosomes ( Figure 4a), indicating the loss of the DAX-1 gene as the cause of AHC in this family.
  • Nucleotide 1292 of the coding sequence representing the guanine in the second position of the serine codon (AGT) at residue 431 in the deduced amino acid sequence (S431), was deleted, resulting in a frameshift and premature termination of transcription.
  • AGT serine codon
  • S431 deduced amino acid sequence
  • a third family containing two brothers diagnosed with AHC associated with mental retardation, showed a larger deletion, extending in a telomeric direction from a centromeric breakpoint between the AHC and GK genes. Larger deletions extending telomerically from presumed centromeric breakpoints within the AHC locus or between the AHC and GK loci have been observed in two boys with isolated AHC and mental retardation.
  • Diagnosis of adrenal insufficiency in order to initiate glucocorticoid and mineralocorticoid replacement therapy, is possible with standard endocrinologic testing, and does not require molecular genetic methods.
  • appropriate management of the associated problems requires a specific diagnosis of the cause of the adrenal insufficiency.
  • Hypogonadotropic hypogonadism HH
  • the DAX-1 cDNA and genomic reagents facilitate the specific diagnosis of AHC, and, therefore, predicts HH in the prepubertal period.
  • a specific molecular genetic diagnosis also will allow more accurate genetic counseling of families with X-linked AHC.
  • the ligand for the DAX-1 nuclear receptor protein remains to be defined, but it is clear that mutations in this new X-linked orphan receptor have significant consequences for adrenal and gonadal development.
  • DAX-1 shows sequence similarity with the nuclear hormone receptor superfamily, the highest similarities being observed with the RXR and orphan (COUP, ARP-1 and EAR-2) subfamilies.
  • the DAX-1 protein binds DNA and is localized to the nucleus.
  • the DAX-1 protein does not contain a recognized canonical zinc finger DNA binding domain, the protein contains cysteine residues that are situated appropriately for interaction with a zinc ion. Therefore we would propose that the DAX-1 gene encodes a new, putative zinc finger protein that is involved in the transcriptional regulation of genes involved in the normal development of adrenal function and secondary sex characteristics.
  • Figure 5 shows the amino acid sequence in the putative zinc finger domain of the DAX-1 protein. The regions of DAX-1 that contain the putative zinc fingers are shown. We have drawn the interaction of the zinc ion with cysteines 41 and 107, but cannot rule out the alternative possibility of an interaction with cysteines 43 and 109.
  • Nucleotide sequence analysis of the cloned genomic sequence utilized the BLAST E-Mail Server at National Center of Biotechnology Information. Computerized assessment of intron-exon boundaries employed the CodonPreference and Testcode programs, and evaluation of the nucleotide and deduced amino acid sequences relied on the GCG package.
  • SSCP Single-strand conformational polymorphism
  • the 50 ⁇ l PCR reaction contained 200ng genomic DNA, the appropriate primers (20pM each), 20nM dNTP, 0.1 ml ⁇ -32P-dCTP (3000Ci/mMol and lOmCi/ml; Amersham, Arlington Heights IL) and the PCR buffer contained lOmM Tris-HCl (pH8.8), 50mM KCl, 1.5mM MgC12, and 0.1 % gelatin. Two units of Taq DNA polymerase (Perkin Elmer) were added at 80oC after a 5 min. hot start at 94oC.
  • the PCR amplification conditions were 0.5 min denamration at 94oC, 0.5 min annealing at 55oC and 1.0 min extension at 72oC for 30 cycles.
  • a 3 ⁇ l aliquot of the PCR reaction was mixed with 9 ⁇ l of the stop solution (95% formamide, lOmM EDTA pH8.0, 0.01 % xylene cyanole) and denamred for 5 min at 94oC.
  • Figure 6 shows the locations of the SSCP fragments in the DAX-1 gene.
  • the bold numbers indicate the orientation of the 8851 bp genomic sequence from nt 1 (on the left) to nt 8851 (on the right).
  • the numbers for the 10 SSCP fragments are keyed to those in Table 1.
  • PCR amplification of DNA The 258 bp fragment containing the tetra-nucleotide tandem repeat was amplified from the cosmid 8E10 13 clone which contain the entire DAX-1 gene, using primers 15 (5 ' -ACCAGCTGATACAGAATCAT-3 ') and 3140 (5'-CAATTCCAGGTCCTGGAGAA-3').
  • the PCR conditions for amplification of this repeats fragment were 1.0 min. denamration at 94oC, 1.0 min. annealing at 57oC, and 1.0 min. extension at 72oC for 32 cycles.
  • the 266 bp sequence ( Figure 6) from the second exon of the gene which detected the 2 bp insertion was amplified from genomic DNA of an AHC patient using primers 3107 (5'-GCTAGCAAAGGACTCTGTGGTG-3') and 2851 (5--TCCATGCTGACTGTGCCGAT-3').
  • the 201 bp fragment ( Figure 6) from the first exon of the gene which showed a nucleotide substimtion was amplified from the genomic DNA from two AHC patients and one carrier from two generations of the same family and also showed a 2 bp deletion in one AHC patient from a different family using primers 3557 (5 '-GTCGCCTCGGGCGCCTTCGG-3 ') and 3603 (5 '-CTGGTGGATCAGTGTTGGGGCT-3 ').
  • the 225 bp fragment ( Figure 6) amplification product, from the first exon of the DNA from an AHC patient showed a nucleotide substimtion polymorphism in many individuals including AHC patients and normal controls using primers 3544 (5 '-GCTTGCTCACTAGCTCAAAGC-3 ') and 3600
  • the membrane was hybridized with a 1.6 kb Sacl genomic fragment which contains the first exon of the AHC gene.
  • the probe was labeled with a-32P dCTP using the random hexamer primer labeling method, and was preassociated with 8-10 mg/ml sheared human placental DNA in 5 X SSPE, 10 X Denhardts, and 2% SDS for 3 hours.
  • the hybridization was carried out at 65 oC for 18-20 hours. Following hybridization, the blot was washed in 2 X SSC, and 0.05% SDS at room temperamre for up to 20 min. A second wash in 2 X SSC, and 0.1 % SDS was performed for up to 15 min. at 65oC 18.
  • Complete sequencing of the DAX-1 gene The DAX-1 genomic region that was completely sequenced was contained within three EcoRI fragments (3.4, 1.4 and 4 Kb ordered from centromere to telomere). Two open reading frames were identified, and, when compared with the cDNA sequence, represented 1168 bp of coding sequence for first exon, a 3385 bp intron and a 245 bp coding sequence for the second exon. The complete sequence for the three EcoRI restriction fragments was 8851 bp in length and was deposited in GenBank (accession no. U31929).
  • Figure 12 shows the nucleotide and predicted amino-acid sequence of DAX- 1. The position of the intron and the sequence of the splice sites is shown above the sequence. Three potential polyadenylation signals were identified in the 3' noncoding sequence of the DAX-1 gene (underlined). The second of these signals (shown in bold) is used in the only polyadenylated cDNA we isolated. The arrow corresponds to the site of polyadenylation. The horizontal arrows correspond to the oligonucleotide primers used to analyse AHC patients. Exon/intron organization of the gene.
  • the first nucleotide of the presumptive initiation codon for the first exon was at position 1580 in the 3.4 kb EcoRI fragment, and the exon ended at position 2747. The rest of this fragment and the 1.4kb EcoRI fragment contained intronic sequence. The remainder of the intron and second exon were located in the 4 kb EcoRI fragment.
  • the coding region of the second exon consisted of a total of 245 base pairs starting at position 6133 with the third nucleotide of the stop codon at position 6377.
  • the intron of the DAX-1 gene was 3385 bp in size and showed a highly conserved splice site at the exon-intron boundary with a GT at the donor site and an AG at the acceptor site ( Figure 7A).
  • Figure 7a shows the genomic organization of the DAX-1 gene.
  • the two exons of the DAX-1 gene are shown as boxed regions with numbers on the sides indicating the size of each exon.
  • the 5' and 3' untranslated region and the intron are represented by the solid line and the numbers undemeath the lines indicate the size of each region.
  • the numbers at the beginning and the end of the solid line indicate the total size of genomic region which has been sequenced, 8851 bp.
  • the conserved splice sites are indicated as bolded letters and a short portion of the intronic sequence is represented by the splice junction regions.
  • the box at the bottom shows the putative DNA binding domain indicated by the arrows in the box, and the ligand binding domain indicated by the open box.
  • Figure 7b shows the nucleotide sequence of a portion of exon 1 and the 5'-flanking region.
  • the putative TATA box, GC boxes and the SF-1 response element are underlined and the GGAA tetra-nucleotide tandem repeat [(GGAA)6GGAAA(GGAA)5GGAAAGGAAGGAAA(GGAA)10] is boxed.
  • the numbering of the nucleotide sequence on the right is relative to the transcription start site.
  • Putative promoter and 3' untranslated region of DAX-1 The 1579 bp of sequence upstream of the transcription initiation site was examined for distinguishing promoter elements.
  • a putative TATA box was identified at bases -58 to -53 upstream of first nucleotide of the initiation codon and a putative steroidogenic factor 1 (SF-1) response element (CCGAGGTCA) was present between bases -172 to -164, relative to the translation start site.
  • the promoter region of the DAX-1 gene was found to be abundant in G+C content. Another prominent feamre was the existence of several GC boxes, GGGCGG (CCGCCC), the recognition site of the Spl transcription factor.
  • GC elements are located at positions -167, -110 and + 154 ( Figure 7B).
  • Two AAT AAA potential polyadenylation recognition sites were identified at positions 6427 and 6466. The second one was the one used as the polyadenylation site in the cDNA clone that we identified previously.
  • Figure 8a shows alignment of the C-terminal ligand binding domain of several members of the nuclear hormone receptors, using DAX-1 as the basis for comparison. The lower case prefixed letters refer to the species from which the gene was obtained: m, mouse; r, rat; and h, human.
  • TR thyroid hormone receptor
  • COUP-TF1 chicken ovalbumin upstream promoter transcription factor
  • RAR retinoic acid receptor
  • RXR retinoic X receptor
  • ERR estrogen related receptor
  • SF-1 steroidogenesis factor-l
  • HNF hepatocyte nuclear factor
  • GCNF germ cell nuclear factor
  • VDR vitamin D3 receptor
  • MR mineralocorticoid receptor
  • GR glucocoticoid receptor
  • PR progesterone receptor
  • AR androgen receptor
  • ER estrogen receptor.
  • the numbers on each side of the bars represent the beginning and end of the amino acid positions used in the comparison to DAX-1 for each member.
  • the gray and hatched boxes represent conserved regions II and IH in the nuclear hormone receptors, respectively.
  • Figures 8b and 8c show amino acid alignment of the conserved regions II and III of same group of the nuclear hormone receptors. Amino acids identical to DAX-1 are represented with a dash. Similar amino acids are bolded. The numbers on the right represent the positions of amino acid residues in the individual receptors. In region II the identity ranges from 24 to 36% and the similarity ranges from 43 to 60%, and in region III the identity ranges from 22 to 57% and similarity ranges from 23 to 61 % .
  • Figure 9 shows polymorphisms and mutations in DAX-1 gene.
  • GGAA tetra-nucleotide tandem repeat polymorphisms Figure 9a.
  • Samples 6 and 15 are from female individuals and the rest are from males.
  • GGAA tetra-nucleotide tandem repeat in an AHC affected family shows Mendelian inheritance of the polymorphism ( Figure 9b).
  • the 266bp fragment of DNA was amplified with primers 3107 and 2851 and sequenced ( Figure 9c).
  • Patient DT shows an additional two cytosine residues compared with control. The arrow points to the normal control without these two additional cytosines.
  • the 201bp fragment of DNA was amplified with primers 3557 and 3603 and sequenced ( Figure 9d).
  • the arrow points to the two nucleotides (AG) which are deleted in patient JLB.
  • the 201bp fragment of DNA was amplified with primers 3557 and 3603 and sequenced ( Figure 9e).
  • the arrow points to the mutation in the patient's DNA sequence, resulting in TAA instead of normal control sequence of TAC.
  • DNA samples from male patients with isolated AHC were screened by SSCP and regions with altered SSCP patterns were sequenced.
  • a 2 bp insertion of cytosine nucleotides was noted between nt. positions 6231 and 6232 in the normal sequence of the second exon ( Figure 9C) and in DNA from patient JLB a 2 bp deletion (AG) was identified between nt. 1733 to 1734 of the normal sequence for the first exon ( Figure 9D), both mutations causing the frame shift and pre-mature termination.
  • a transversion at position 1852, which changed codon from TAC (Tyr) to TAA (stop) (Y91X) in the first exon of the gene was detected in two AHC patients, PS and RH, who were from different generations of the same family ( Figure 9E).
  • a nucleotide transition at position 2077 changed codon 166 from CGG to CGA in the first exon, and was found in several isolated AHC patients and in normal controls. Since CGG and CGA encode the same amino acid, arginine, this nucleotide change represented a polymorphism.
  • Interspecies zoo blot analysis An interspecies zoo blot of EcoRI digested genomic DNA from several species was analyzed.
  • the genomic DNA samples from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast were digested by EcoRI and hybridized with a DAX-1 cDNA probe from the 1.6 kb Sacl fragment which contains entire first exon of the DAX-1 gene.
  • the numbers on the left side of the blot represent the sizes of the markers ( Figure 10).
  • DAX-1 genomic strucmre of the DAX-1 gene by sequencing three EcoRI restriction fragments totaling more than 8.8 kb. These fragments had been previously shown to be contained in a cosmid used to clone the DAX-1 cDNA by a genomic scanning strategy. Sequencing of these DNA fragments showed that the DAX-1 gene consists of two exons separated by a 3.4 kb intron. The promoter region of the gene contains a putative SF-1 response element, as well as a putative TATA box and several Spl sites. Knowledge of the genomic sequence permitted us to develop an SSCP strategy for rapid mutation detection, and three new mutations were identified.
  • Polymorphisms including sequence tandem repeats, that may be useful for linkage analysis in families with an affected individual, but no known mutations, have also been identified in the DAX-1 gene.
  • An interspecies zoo blot indicates that there may be a DAX-1 related gene in the yeast genome.
  • the CCGAGGTCA sequence which is located 175 bases upstream of the initiation codon, is a putative SF-1 response element.
  • SF-1 is a member of the nuclear hormone receptor superfamily that regulates the expression of many steroidogenic enzymes, such as cholesterol side chain cleavage enzyme (SCC), steroid 21 -hydroxylase, and the aldosterone synthase isozyme of the steroid beta-hydroxylase.
  • SCC cholesterol side chain cleavage enzyme
  • steroid 21 -hydroxylase steroid 21 -hydroxylase
  • aldosterone synthase aldosterone synthase isozyme of the steroid beta-hydroxylase.
  • SCC cholesterol side chain cleavage enzyme
  • steroid 21 -hydroxylase steroid 21 -hydroxylase
  • aldosterone synthase aldosterone synthase isozyme of the steroid beta-hydroxylase.
  • SCC cholesterol side chain cleavage enzyme
  • All of these steroidogenic hydroxylases have SF-1 responsive elements in the promoter region of the genes, with AGGTCA as core sequence. Disruption of the Ftz-Fl gene encoding SF-1 in the mouse precludes adrenal and gonadal development. In mice homozygous for a null mutation in the SF-1 locus, the adrenal glands and gonads fail to develop and the mice die of adrenal insufficiency. AHC patients who have deletions involving the DAX-1 gene or a mutation within this gene also show a phenotype with failure of the adrenal cortex to develop normally and adrenal insufficiency. Interestingly, both SF-1 and DAX-1 are expressed in many of the same tissues such as the adrenal cortex, testis and ovaries.
  • the DNA binding domain of the nuclear hormone receptor superfamily usually contains 66 amino acids and is rich in lysine, arginine and cysteine residues. This domain is highly conserved among superfamily members and a number of residues are invariantly conserved throughout evolution. The positions of the key cysteine residues are absolutely conserved yielding a motif of two C2C2 domains: CXXC...X7...H..X5.. CXXC..X15...C..X5..C...X9...CXXC..X4..C.
  • each C2C2 strucmre chelates a single Zn2 + ion forming a strucmre termed a zinc finger.
  • a zinc finger Within the putative DNA binding domain in the N-terminus of DAX-1, an unusual zinc finger motif has been identified, containing two C2C2 zinc fmgers (CXXCXC...X22...CXXC) within three and half tandem repeats of a 65 to 67 amino acid motif.
  • the DAX-1 zinc finger appears to be unique, it is consistent with the other DNA binding domain structural motifs in other superfamily members.
  • Figure 11 is a comparison of zinc coordinating domains of DNA binding proteins.
  • the single letter designations are: C, Cys; H, His; and X is any amino acid. Subscripted letters represent the numbers of the amino acids in the segment.
  • the DNA binding domain of all nuclear hormone receptors cloned to date are encoded by genes containing several introns except for COUP-TF.
  • the DNA binding domain of COUP-TF is encoded by a gene with a single intron and it has been suggested that COUP-TF is an ancestral receptor based on the simple genomic organization. DAX-1 also shows this simple organizational pattem.
  • the members of the nuclear receptor gene superfamily are identified by the three conserved structural features that separate these proteins from other transcription factor families.
  • the conserved DNA binding domain is designated region I and regions II and III are located in the ligand binding domain.
  • the functions of regions II and III are still unknown, but may involve dimerization, transactivation or transrepression along with ligand binding.
  • DAX-1 coding sequence only two mutations have been observed that alter single amino acids.
  • both of these mutations are within the conserved region II in the ligand binding domain.
  • the ligand binding domain of DAX-1 appears indispensable for the function of the protein. It has been demonstrated that many if not all nuclear hormone receptors are functional in yeast. This suggests that the machinery required for nuclear receptor transcriptional function is conserved from yeast to mammals. This has been surprising since no nuclear receptor-like proteins have been identified in yeast. However, our data suggest the existence of a DAX-1-like gene in yeast. Thus, the components of the transcriptional machinery required to interact with the ligand binding domain of DAX-1 or a DAX-1-like protein may be normally expressed in yeast, and these same components may be utilized by heterologously expressed mammalian nuclear receptors.
  • the nuclear receptors have been grouped into three subfamilies depending upon the relative conservation of the DNA binding domain.
  • the putative DNA binding domain of the DAX-1 gene is composed of three and half repeats of a 65-67 amino acid motif, which contains two potential novel zinc finger strucmres. This domain shows no similarity to any other nuclear receptor indicating that it defines a new class within the superfamily.
  • This experiment was to determine if DAX-1 was expressed in the nervous system, particularly in the hypothalamus and pimitary, in order to better understand the pathogenesis of the HH that is frequently associated with X-linked AHC.
  • the northern blot contained ⁇ mg of poly A+ RNA per lane from various human tissues.
  • the northern blot was prehybridized in 5 X SSPE, 10 X Denhardts, 2% SDS, and 8-10 ⁇ g/ml sheared human placental DNA, for 3 hours.
  • the probe was labeled with a32P-dCTP using the random hexamer primer labeling method, and was preassociated with 8-10 ⁇ g/ml sheared human placental DNA in 5 X SSPE, 10X Denhardts, and 2% SDS for 3 hours.
  • the hybridization was carried out at 65 °C for 18-20 hours.
  • the blot was washed in 2 X SSC, and 0.05 % SDS at room temperamre for up to 20 min. If required because of excess residual radioactivity, a second wash in 2 X SSC, and 0.1 % SDS was performed for up to 15 min. at 65 °C.
  • a human adrenocortical carcinoma cell line, NCI-H295 was obtained from the American Type Culmre Collection (ATCC; Rockville, MD).
  • the following human cDNA libraries were purchased from Clontech (Palo Alto, CA): fetal adrenal (dgtllHL1118b); hypothalamus (lgtll HL1172B); kidney (lgtl l HL1071b); liver (lgtll HL1115b); and testis (lgtll HLll ⁇ la).
  • Human poly A+ RNA prepared from total adult brain (6516-1), fetal brain (6525-1), pimitary (6584-1) and placenta (6518-1), was purchased from Clontech and was used for RT-PCR preparation of cDNA.
  • RNA isolation Total RNA was isolated from 1x108 NCI-H295 cells by the guanidinium thiocyanate method using Promega RNA AgentsO RNA Isolation Kit (Promega, Madison, WI).
  • RNAsin Reverse transcription - polymerase chain reaction
  • RT-PCR Reverse transcription - polymerase chain reaction
  • the reaction mixmres contained 2 ⁇ l of random hexamer (300 ng/ ⁇ l), 2 ⁇ l of oligo (dT) (300 ng/ ⁇ l), 2 ⁇ l of 20mM DTT, 1.6 ⁇ l of 25 mM dNTP, 4 ⁇ l of lOxbuffer (750 mM KCL, 30mM MgC12 and 500mM TrisHCl pH 8.3, 0.5 ml of RNAsin (Promega, Madison, WI), l ⁇ l of M-MLV (GIBCO BRL, Gaithersburg, MD), and water to bring the total volume to 40 ⁇ l.
  • the reaction was carried out at 70 °C for 5 min. then 37 °C for 90 min.
  • primer pair was selected, such that primer 2937 (5 ' - AAGG AGT ACGCCT ACCTC AA-3 * ) was located in the first exon of the
  • DAX-1 gene and 2851 (5'-TCCATGCTGACTGTGCCGAT-3') was located in the second exon; therefore, any contamination from genomic DNA or unprocessed RNA would have been detectable by its larger size.
  • Amplification conditions were as follows: 5 min at 95 °C without Taq polymerase, then 0.5 min. denamration at 94°C, 0.5 min. annealing at 57°C and 0.5 min. extension at 72°C for 32 cycles using Taq polymerase (Boehringer Mannheim) and amplification buffer (Perkin Elmer).
  • the probe was labeled with 32P- dCTP, using the random hexamer primer labeling method, and was preassociated with 8-10 mg/ml sheared human placental DNA in 5 X SSPE, 10 X Denhardt's, and 2% SDS for 3 hours.
  • the hybridization was carried out at 65 °C for 18-20 hours.
  • the blot was washed in 2 X SSC and 0.05 % SDS at room temperamre for up to 20 min. A second wash in 2 X SSC and 0.1 % SDS was performed for up to 15 min. at 65 °C.
  • RNA from a variety of human tissues was hybridized with the Sad restriction fragment containing the first exon of the DAX-1 gene. The results show that the gene is strongly expressed in testis and weakly expressed in ovary. Two different RNA species were observed, one approximately 1.9 kb in length and the other about 6 kb in size. The 1.9 kb band was consistent with the size of the cDNA. The 6kb band, but not the 1.9 kb band, hybridized with a probe containing only intronic sequence from the DAX-1 gene, indicating that the 6 kb band represented unprocessed RNA.
  • DAX-1 expressed in human hypothalamus, pimitary gland and an adrenocortical carcinomal cell line RT- PCR amplified a 251 bp DAX-1 sequence from mRNA and cDNA libraries.
  • RNA unprocessed RNA in the Northern blot, only the smaller amplification product from processed mRNA was observed, presumably due to reduced efficiency of amplification of the longer sequence containing the intron.
  • RT-PCR amplified the 251 bp DAX-1 product from mRNA extracted from total adult brain, total fetal brain, pimitary gland, and the human adrenocortical carcinoma cell line, NCI-H295, as well as from cDNA libraries of the hypothalamus, fetal adrenal gland, and testis.
  • the DAX-1 product was negative in mRNA from placental tissue and cDNA libraries from liver and kidney.
  • a Southern blot was prepared from the gel. Placenta, liver and kidney remained negative for DAX-1.
  • DAX-1 is not only expressed in mRNA from adrenal and gonadal tissues, but also from adult and fetal brain. More specifically, DAX-1 is expressed in the hypothalamic-pituitary axis.
  • DAX-1 is expressed in a human adrenocortical carcinoma cell line, NCI- H295, that is a steroidogenic model for the human fetal cortex , and expresses most of the enzymes associated with adrenal steroidogenesis.
  • the genes encoding the steroidogenic enzymes in this cell line respond to stimulation by second messenger pathways in a manner similar to the human adrenal cortex. Investigation of this cell line will help us to understand the regulation of DAX-1 expression, and the role of DAX-1 in the fetal adrenal.
  • DAX-1 may be a useful marker for mmor cell transformation in the specific tissues of the hypothalamic pituitary-adrenal/gonadal axis.
  • the development of the mammalian reproductive system requires elaborate interactions within the hypothalamic-pimitary-adrenal/gonadal axis.
  • the gonadal steroids, essential reproductive hormones are produced by testes and ovaries under the regulation of the tropic hormones LH and FSH.
  • LH and FSH The release of LH and FSH by die gonadotropes of the anterior pimitary gland is stimulated by GnRH, which is secreted by specific neurons located within the hypothalamus.
  • GnRH which is secreted by specific neurons located within the hypothalamus.
  • a number of neuronal centers deliver the modulatory inputs to GnRH neurons that transduce the elaborate effects of behavioral and environmental stimuli on the reproductive axis. All of these complex interactions are modulated at both the hypothalamic and pimitary levels through negative and positive feedback regulation.
  • DAX-1 may be involved with the normal development of GnRH neurons and gonadotropes or the regulation of GnRH and gonadotropin expression. Since pulsatile administration of GnRH to males with HH associated with AHC can result in testosterone secretion, the action of DAX-1 would appear to be proximal to GnRH in the hypothalamus and/or pimitary.
  • DAX-1 is a orphan nuclear receptor that is an essential regulator of steroid hydroxylase gene expression, and is expressed at multiple levels of the reproductive system from hypothalamus and pimitary to the adrenal cortex and gonads. SF-1 has been demonstrated to be essential for development of the adrenal cortex, gonads and the ventromedial nucleus of the hypothalamus.
  • the promoter region of the DAX-1 gene contains a putative SF-1 response element and now we show that DAX-1 is expressed in hypothalamic-pimitary-adrenal/gonadal axis.
  • DAX-1 may be regulated by SF-1 or may act in concert with SF-1 as a co-regulator at various levels in gonadal and adrenal development.
  • JK2 genomic DNA fragment which is a genetic marker telomeric to the GK locus and most centromeric to the AHC locus, was used to screen a human chromosome X cosmid library (Guo et al., 1995). Two positive clones were identified from this library and the end fragments of these two positive clones were used for chromosome walking in the telomeric portion of this region within Xp21. The same strategy was repeated until a cosmid contig covered the region between the marker YHX133R and K23-b2, a total of approximately 160 kb.
  • Figure 14 shows relative locations of the genes responsible for cytomegalic adrenal hypoplasia congenita (AHC), glycerol kinase deficiency (GK), and Duchenne's muscular dystrophy (DMD) within the X chromosome.
  • AHC cytomegalic adrenal hypoplasia congenita
  • GK glycerol kinase deficiency
  • DMD Duchenne's muscular dystrophy
  • Figure 15 shows positional cloning of the DAX-1 gene in Xp21.
  • the end of the short arm of the X chromosome is to the left of the figure (Xp-ter) and the centromere is to the right (Xp-cen).
  • the markers within the region are indicated along the X chromosome.
  • the cosmid contig is illustrated below the X-chromosome and the cosmids containing at least a fragment of the DAX-1 gene are named.
  • the genomic strucmre of the DAX-1 gene is illustrated at the bottom of the figure. (Adapted from Guo et al., 1995)
  • the cosmid clones in the contig were digested with EcoRI and hybridized with a polymerase chain reaction (PCR) amplified radiolabeled human fetal adrenal cDNA library.
  • PCR polymerase chain reaction
  • Two bands (4 kb and 3.4 kb) were positive in four of these clones (8E10, 38A2, 38C4, 230H3) and one band (3.4 kb) was positive in one of these clones (17G12) ( Figure 16). Both fragments were subcloned and sequenced. An open reading frame was identified in each of these two fragments.
  • Figure 16 shows results of a Southern blot of a restriction digest from contig cosmids hybridized with the PCR amplified, labeled human fetal adrenal cDNA library.
  • the hybridizing bands in cosmid 8E10, 38C4 and 230H3 were 3.4 kb and 4 kb and the hybridizing band in cosmid 17G12 was 3.4 kb.
  • the 3.4 kb fragment was used to screen two human fetal adrenal cDNA libraries and eight positive clones were identified. After sequencing the eight cDNA clones, it was determined that their sequences were identical to the open reading frame of the genomic fragment.
  • the candidate gene responsible for AHC consists of two exons separated by a 3.4 kb intron ( Figure 17). It was determined that both the 4.0 kb and 3.4 kb fragments identified with the CAIGES method were fragments of the same gene with exon 1 contained in the 4.0 kb fragment and exon 2 in the 3.4 kb fragment. This gene was named DAX-1 based on its localization within the dosage sensitive sex reversal (DSS) locus and the AHC locus on the X chromosome (Zanaria et al, 1994).
  • DSS dosage sensitive sex reversal
  • Figure 17 shows the genomic strucmre of the DAX-1 gene. Boxed strucmres represent the exons and include the relative positions of domains within the DAX-1 protein.
  • the R domain is the unique amino-terminal domain which contains the repeated motifs and the two putative zinc fingers.
  • the E domain is the ligand binding domain. Regions II and III are highly conserved regions defmed within the ligand binding domain of all nuclear hormone receptors (Wang et al., 1989).
  • the initiation codon, splice junctions, and termination codon are indicated.
  • Other sequences of interest including the TATA box, putative steroidogenic factor 1 response element, polyadenylation sites, and polymorphic sequences are indicated.
  • the cDNA sequence of this gene contains an open reading frame of 1410 bp, predicting a protein of 470 amino acids.
  • Southern blot analysis of patients with AHC deletions provides strong indication for the involvement of the DAX-1 gene in X-linked AHC (Zanaria et al, 1994; Muscatelli et al, 1994; Guo et al, 1995).
  • the cDNA clone was hybridized to genomic DNA isolated from AHC patients with deletions. In normal patient controls two bands were identified (4.0 kb and 3.4 kb); however, in AHC deleted patients this normal pattem was not present indicating that patients with AHC lack this gene.
  • the DAX-1 gene was responsible for AHC we focused on patients with isolated AHC. Presumably, AHC in these patients is caused by point mutations or small DNA rearrangements.
  • the DAX-1 gene encodes a member of the nuclear hormone receptor superfamily based on the presence of a conserved ligand binding domain present in its carboxy-terminus.
  • Members of the nuclear hormone receptor superfamily are transcription factors that regulate a myriad of essential cellular processes.
  • Many members of this superfamily such as the steroid receptors (i.e. progesterone and glucocorticoid receptors), thyroid hormone receptors, and the receptors for lipophilic vitamins (i.e.
  • retinoic acid and vitamin D3 regulate transcription in a ligand dependent manner
  • a ligand dependent manner Evans, 1988; O'Malley, 1990; Beato, 1991; Green and Chambon, 1988, Baniahmad and Tsai, 1993; Truss and Beato, 1993; Baniahmad et al, 1994.
  • the orphan receptors which have a not yet identified ligand or act in a ligand independent manner (O'Malley, 1990; O'Malley and Conneely, 1992; Power et al, 1992; Lydon et al., 1992; Conneely and O'Malley, 1994).
  • A/B region is the most amino terminal region and is least conserved between the receptors.
  • Some receptors such as the progesterone or glucocorticoid receptors, have A/B regions that are several hundred amino acids in length, while in some orphan receptors this region consists of only a few amino acids.
  • Region C is the most highly conserved region and contains the DNA binding domain (DBD) which contains two zinc fingers (Cooney and Tsai, 1994; Glass, 1994). This highly conserved domain has also been termed region I (Wang et al., 1989).
  • Region D known as the hinge region, links the DBD to the ligand binding domain (LBD). This area is not well characterized, but it may contain a transactivation domain and regions required for transcriptional silencing in the thyroid hormone and retinoic acid receptors (Baniahmad et al., 1992; Baniahmad et al., 1993; Baniahmad et al. 1995).
  • Region E consists of the LBD, is contained in the carboxy-terminus, and is the second most conserved domain between family members. This region also contains many overlapping functional domains, including those required for nuclear localization, dimerization, and transactivation (Webster et al., 1988; Kumar et al., 1986; Picard et al., 1988; Guiochon-Mantel et al., 1989; Kumar and Chambon, 1988; Fawell et al., 1990; Lee et al., 1992). Two areas within the LBD have been identified which have a very high level of conservation and these have been termed regions II and III (Wang et al., 1989).
  • region DT consists of helix 8 and the "loop" of amino acids just amino-terminal to this helix (Bourguet et al., 1995).
  • the "loop" of amino acids between helices 7 and 8 has been shown to be a component of the interface between a RXR ligand binding domain homodimer (Bourguet et al., 1995).
  • region HI seems to play an important role in dimerization by serving as a component of the dimerization interface.
  • region F The most carboxy-terminal region within nuclear hormone receptors is region F and is present in only a few family members such as RAR and ER. The function of this region is unclear.
  • DAX-1 is a very unusual member of the nuclear hormone receptor superfamily in that it does not have the typical A-F region structure.
  • Figure 18 shows a comparison of the domain strucmre of DAX-1 to a prototypic member of the nuclear hormone receptor superfamily.
  • the A through E domain strucmre of a nuclear receptor (Krust et al., 1986) is illustrated along with the R-E domain strucmre of DAX-1.
  • the R domain is the unique amino-terminal domain which contains the repeated motifs and the two putative zinc fingers.
  • the ligand binding domains (LBD) are aligned with the relative locations of regions II and III (Wang et al., 1989).
  • the relative location of the DNA binding domain (DBD) of the nuclear receptor is also illustrated.
  • DAX-1 does not contain regions A through D, but has been classified as a member of the nuclear hormone receptor superfamily based on the presence of a highly conserved region E. Within the DAX-1 region E are the two well conserved regions II and III defined for classical members of the superfamily ( Figure 18). The highest similarities in amino acid sequence were observed with RAR, RXR, COUP, and TR2-11 and the similarities ranged from 42 to 52% . Analysis of the DAX-1 carboxy-terminus revealed that it contains an entire LBD with highly conserved regions II and III.
  • FIG. 19 and 20 Alignments of regions II and IH within the LBD between DAX-1 and several other members of the nuclear hormone receptor superfamily are shown in Figures 19 and 20.
  • the percentage of identical amino acids in region II ranges from 24 to 36% and in region III from 22 to 57% .
  • the ranges of identical amino acids within the LBD regions is common for superfamily members containing the classical A-F domain strucmre.
  • DAX-1 also contains the heptad repeats which are conserved in the nuclear hormone receptor superfamily (Zanaria et al., 1994). The heptad repeats have been proposed to play a role in the interface between receptor dimers (Forman and Samuels, 1990).
  • Figure 19 shows a comparison of the ligand binding domain of DAX-1 to other members of the nuclear hormone receptor superfamily.
  • the numbers within the boxed regions II and III indicate the percentage of identical amino acids within these regions.
  • Figure 20 shows alignment of the amino acids within regions II and III.
  • This transactivation domain has been identified and characterized in many receptors including RXR, RAR, ER, and TR (Durand et al., 1994; Tate et al., 1994; Danielian et al., 1992; Baniahmad et al., 1995; Leng et al., 1995).
  • the carboxy-terminal region of DAX-1 contains a conserved LBD, its amino-terminus is very unusual and we have proposed that this strucmre may be a novel DNA binding domain (Zanaria et al., 1994; Guo et al., 1995).
  • the amino-terminal region is very rich in small amino acids such as glycine and alanine and is arranged in 3.5 tandem repeats of a 65-67 amino acid motif with cysteines in conserved positions ( Figure 22).
  • Figure 21 shows a comparison of the AF-2 or t4 transactivation domain of DAX-1 to other nuclear hormone receptors.
  • the conserved hydrophobic regions are boxed and the invariant glutamate residue is outlined.
  • Asterisks represent the carboxy-terminus of the receptor. Numbers to the right of the alignment indicate the amino acid position within the receptor.
  • Figure 22 shows that the putative DNA binding domain of DAX-1 is composed of 3.5 repeats of a 65-67 amino acid motif containing two zinc finger like strucmres.
  • Figure 22a shows alignment of the 3.5 repeats within the amino-terminus of D AX- 1. Dashed lines indicate identical amino acids. Shaded boxes indicate the position of the conserved cysteines. Numbers to the right reflect the amino acid number within the DAX-1 coding sequence. Dots represent gaps inserted into the sequence during the alignment.
  • Figure 22b shows a comparison of the putative DAX-1 zinc finger to zinc fingers found in other double stranded DNA binding proteins.
  • Figure 23 is a schematic of the DAX-1 protein illustrating the approximate location of various types of mutations causing AHC. Below the schematic is a Table containing all of the known mutations in the DAX-1 gene. The namre of each mutation is indicated. The numbers shown on the schematic correspond to the numbered mutations in the Table.
  • DAX-1 is the first identified member of a novel subfamily of the nuclear hormone receptor superfamily which contains this relatively unusual domain organization; whether this is the sole member of this subfamily or other members will be defined remains to be determined.
  • DAX-1 is expressed in the fetal adrenal gland (Zanaria et al., 1994; Guo et al., 1995).
  • DAX-1 is also expressed in the adult adrenal glands, although at a much lower level than in the fetal gland (Guo et al., in press). Interestingly, DAX-1 is also expressed in the ovaries and testes, suggesting that DAX-1 may play a role in the regulation of steroidogenesis (Zanaria et al.). As a putative transcription factor expressed in steroidogenic tissues, DAX-1 is a candidate gene for the sex reversal locus, since DAX-1 is contained in the DSS region (Bardoni et al., 1994).
  • DAX-1 is also expressed in both the hypothalamus and pimitary gland; thus, DAX-1 is expressed at each level of the hypothalamic-pimitary-adrenal/gonadal axis.
  • the expression of DAX-1 in the hypothalamus and pimitary gland suggests that interruption of the neuroendocrine expression of DAX-1 may be the cause of the HH that is often associated with AHC (Guo et al., 1995).
  • DAX-1 steroidogenic factor-l
  • SF-1 steroidogenic factor-l
  • Androgen insensitivity syndrome is an X-linked disorder which results in abnormal development of the sexual development of the male.
  • Multiple classes of mutations have been identified in the gene for the androgen receptor in patients with AIS. Both nonsense and missense mutations have been characterized along with mutations that alter the mRNA splicing of the receptor (McPhaul et al., 1991; Marcelli et al., 1990B; Marcelli et al., 1990A; Marcelli et al., 1991; Brown et al, 1990; Lubahn et al., 1989). Mutations that alter only single amino acids in the DBD and LBD have been shown to result in AIS (Marcelli et al., 1990B; Brown et al., 1990).
  • the DAX-1 gene is frequently deleted in patients with the contiguous gene syndrome, complex glycerol kinase deficiency, involving the loss of the GK locus along with the loci for AHC and /or DMD (McCabe, 1994).
  • Patients with isolated AHC, without clinical features of GK deficiency or DMD, and no detectible deletions have also been described.
  • Intragenic mutations, including microdeletions, insertions, and point mutations have been identified in patients with isolated AHC (Zanaria et al., 1994; Muscatelli et al., 1994; Guo et al., 1995).
  • McKusick VA Mendelian Inheritance in Man - Catalogs of Atuosomal Dominant, Autosomal Recessive, and X-Linked Phenotypes, ed 10. Baltimore:The Johns Hopkins University Press; 1992.
  • Hay ID Smail PJ, Forsyth CC. Familial cytomegalic adrenocortical hypoplasia: an X-linked syndrome of pubertal failure. Arch Dis Child. 1981;56:715-721.
  • Zanaria E Muscatelli F, Bardoni B, et al. An unusual member of the nuclear hormone receptor superfamily responsible for x-linked adrenal hypoplasia congentia. Namre. 1994;372:635-641. 16. Muscatelli F, Strom TM, Walker AP, et al. Mutations in the DAX-1 gene give rise to both x-linked adrenal hypoplasia congentia and hypogonadotropic hypogonadism. Namre. 1994;372:672-676.
  • GCG Genetics Computer Group
  • Luo X Ikeda Y and Parker KL 1994 A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77: 481-490.
  • mouse steroidogenic factor 1 an essential regulator of the steroid hydroxylases. Mol. Endocrinol. 8: 654-652.
  • Zanaria E Muscatelli F, Bardoni B, Strom TM, Guioli S, Guo W, Lalli E, Moser C, Walker AP, McCabe ERB, Meitinger T, Monaco AP, Sassone-Corsi P, Camerino G.
  • Luo X Ikeda Y, Parker KL.
  • a cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77:481-490, 1994.
  • Gazder AF Oie HK, Shackleton CH, Chen TR, Triche TJ, Myers CE, Chrousos GP, Brennan MF, Stein CA, La-Rocca RV. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res 50:5488-5496, 1990.

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Abstract

L'invention porte sur une molécule de la protéine DAX-1 possédant une séquence d'acides aminés débutant par la méthionine en position 1 et s'achevant avec l'isoleucine en position 470 comme le montre la figure 12. Cette invention a également trait à une séquence génomique d'acides aminés pour DAX-1, y compris un intron, des exons et une région promoteur. Elle concerne, de surcroît, des procédés d'utilisation et de production de la protéine DAX-1 ainsi que de molécules d'acide nucléique de DAX-1.
PCT/US1996/012310 1995-07-28 1996-07-26 Proteine dax-1, procedes de production et d'utilisation WO1997004810A1 (fr)

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WO2005114179A1 (fr) * 2004-05-13 2005-12-01 Bayer Healthcare Ag Diagnostic et therapie destines aux maladies associees a une sous-famille 0 de recepteurs nucleaires, groupe b, element 1 (nr0b1)
CN107206087A (zh) * 2014-09-19 2017-09-26 瓦伦西亚普林西比菲利普中心团体投资基金会 X连锁肾上腺脑白质营养不良的治疗中的特异性mtor抑制剂

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US7150971B2 (en) * 2001-05-21 2006-12-19 The Regents Of The University Of California Membrane-resident steroid receptors and methods of use thereof
WO2005071418A2 (fr) * 2003-10-29 2005-08-04 Evotec Neurosciences Gmbh Utilisation diagnostique et therapeutique du gene humain dax-1 et proteine pour maladies neurodegeneratives
CA3061361A1 (fr) * 2017-04-25 2018-11-01 Theralase Biotech Inc. Procede et appareil de photoactivation de recepteurs nucleaires
ES2954867A1 (es) * 2022-04-19 2023-11-27 Inst De Salud Carlos Iii Acido nucleico y su uso en el tratamiento del sarcoma de ewing

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US5217867A (en) 1988-11-30 1993-06-08 The Salk Institute For Biological Studies Receptors: their identification, characterization, preparation and use

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND MOLECULAR MEDICINE, Volume 56, issued 1995, GUO et al., "Expression of DAX-1, the Gene Responsible for X-Linked Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism, in the Hypothalamic-Pituitary-Adrenal/Gonadal Axis", pages 8-13. *
JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION, Volume 274, Number 04, issued 26 July 1995, GUO et al., "Diagnosis of X-Linked Adrenal Hypoplasia Congenita by Mutation Analysis of the DAX1 Gene", pages 324-330. *
NATURE, Volume 372, issued 15 December 1994, MUSCATELLI et al., "Mutations in the DAX-1 Gene Give Rise to Both X-Linked Adrenal Hypoplasia Congenita and Hypogonadotropic Hypogonadism", pages 672-676. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005114179A1 (fr) * 2004-05-13 2005-12-01 Bayer Healthcare Ag Diagnostic et therapie destines aux maladies associees a une sous-famille 0 de recepteurs nucleaires, groupe b, element 1 (nr0b1)
CN107206087A (zh) * 2014-09-19 2017-09-26 瓦伦西亚普林西比菲利普中心团体投资基金会 X连锁肾上腺脑白质营养不良的治疗中的特异性mtor抑制剂

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